SptP, a Salmonella typhimurium type III-secreted protein, inhibits the mitogen-activated protein kinase pathway by inhibiting Raf activation

Abstract

SummarySalmonella has developed ways to modulate host cellular response in order to survive. Although the steps required for such modulation have been incompletely characterized, there is increasing evidence for a role for SptP, a type III secretion protein. In part, the actions of SptP are thought to be mediated through its reported inhibition of the extracellular-regulated kinase (ERK) MAP kinase pathway. In the present studies, a series of transfections were performed in which various constitutively activated components of the MAP kinase pathway were co-transfected with SptP in order to determine the mechanism by which SptP inhibits this MAP kinase activation. SptP was found to inhibit the activation of ERK stimulated by both a constitutively active form of Ras and a partially activated form of Raf-1 containing a phospho-mimetic mutation (Raf Y340D). In contrast, the activation of ERK by constitutively active forms of MAP kinase kinase (MEK) was not inhibited, suggesting that the actions of SptP were mediated by Raf-1. In order to determine how SptP might interfere with activation of Raf, we utilized a membrane-localized form of Raf. Constitutive membrane-localization of Raf (Raf-CAAX), resulting in partial activation, did not prevent inhibition by SptP. However, introduction of an additional, partially activating (Y340D) phospho-mimetic mutation, to RafCAAX, dramatically reduced the ability of SptP to inhibit Raf action. Comparison of SptP mutants, lacking either GTPase-activating protein (GAP) activity or tyrosine phosphatase activity, further suggested that SptP inhibits both the membrane localization and subsequent phosphorylation required for activation of Raf. Both tyrosine phosphatase activity and GAP activity were responsible for SptP inhibition of Raf(Y340D)-induced ERK activation, but only GAP activity was responsible for inhibition of the membrane localized forms of Raf-1. To assess the biological significance of SptP, we examined tumour necrosis factor (TNF)-a a a a induction following Salmonella infection. SptP gene deletion enhanced the capacity of Salmonella to induce TNF-a a a a secretion following infection of J774A.1 macrophage cells.

Impact ofSalmonella entericaType III Secretion System Effectors on the Eukaryotic Host Cell

“…This activity is involved in reversing the activation of the MAPK ERK that results from Salmonella infection [31]. SptP inhibits activation of ERK by interfering with activation of Raf1 in a process that involves both SptP activities [303]. Probably as a consequence of downregulation of ERK, SptP contributes, together with SspH1, to downmodulate IL-8 production after invasion of intestinal epithelial cells [304].…”

Molecular Mechanisms Used by Salmonella to Evade the Immune System

“…The C-terminal domain possesses tyrosine phosphatase activity, which is (Kaniga et al, 1996) involved in reversing the initial activation of the MAPK ERK, triggered by Salmonella infection (Murli et al, 2001). SptP inhibits this MAPK pathway through inhibition of Raf1 activation in a process that involves both SptP activities (Lin et al, 2003). This down-modulation of ERK activation could explain the contribution of SptP, together with SspH1, towards the inhibition of IL-8 production after invasion of intestinal epithelial cells (Haraga and Miller, 2003).…”

Salmonella: Invasion, Evasion & Persistence

“…SptP GAP and tyrosine phosphatase activities play critical roles in reversing MAPK activation while AvrA acetyltransferase activity towards MAPK kinases inhibits Jnk activation (Jones et al, 2008;Lin et al, 2003;Murli et al, 2001). Moreover, SpvC has been shown to directly inhibit Erk, Jnk and p38 MAPKs via its phosphothreonine lyase activity (Li et al, 2007;Mazurkiewicz et al, 2008).…”